CELLULAR IMMUNOLOGY 176, 22– 33 (1997) ARTICLE NO. CI961056 Tissue-Specific and Growth-Regulated Expression of CD44 Variable Exon Determinants in the Mouse YUMI YAMASHITA,* ,1 HIROYUKI HIRANO,† AND RICHARD J. HODES* *Experimental Immunology Branch, National Cancer Institute and National Institute on Aging, Bethesda, Maryland 20892; and †Department of Periodontology and Endodontology, Osaka University Faculty of Dentistry, Suita, Osaka 565, Japan Received July 15, 1996; accepted November 5, 1996 interactions which are important to cell differentiation and function (3 – 6). CD44 is a polymorphic transmembrane glycoprotein CD44 is known to exist in a variety of isoforms which widely expressed in lymphocytes and epithelial cells. differ substantially in molecular weight as analyzed in CD44 polymorphism reflects both posttranslational SDS–PAGE gel migration (7 –9). The most abundant modification and alternative splicing of up to 10 vari- ably expressed exons in the membrane-proximal CD44 form, with a molecular weight of 85 – 90 kDa, has been extracellular domain. An analysis of CD44 variable referred to as the ‘‘wild type,’’ standard, or hematopoi- exon-containing isoforms in the mouse was carried out etic form and is the form that is predominantly ex- by generating a panel of monoclonal antibodies pressed on lymphocytes (10, 11). Higher molecular against variable region determinants of CD44. Immu- weight forms, 150–200 kDa, are predominantly ex- nohistochemical analysis demonstrated selective pat- pressed on epithelial cells and exhibit distinct glycosyl- terns of expression of CD44 variable exon determi- ation patterns, including variable modifications with nants in normal tissues, and flow cytometric analysis glycosaminoglycans such as chondroitin sulfate and hep- identified expression of CD44 variable exon-depen- aran sulfate (7, 8). In the past few years, it was found dent determinants in epithelial and lymphoid cell that CD44 isoforms also result from extensive hetero- lines. Regulation of alternative splicing was studied geneity in the protein core sequence as a result of alter- by characterization of cell surface expression of CD44 native splicing among so-called additional, or variable, variable exon determinants on HC11 mammary epithe- exons (8, 12 –14). In mice, 11 of the 21 CD44 exons lial cells, and it was demonstrated that variably have been shown to undergo alternative splicing: one spliced isoforms are selectively regulated as a function variable exon (exon 20) site is located in the cyto- of growth phase in vitro. These results demonstrate plasmic tail and 10 other variably expressed exons can the tissue-specific and growth-regulated expression of be found in a membrane proximal site of the protein’s the CD44 isoform at the level of cell surface protein extracellular domain (15 – 18). products and identify isoform-specific determinants The expression of variable exons by selected CD44 that can be targeted in analysis of isoform-specific isoforms has been associated with a number of interest- function.  1997 Academic Press ing features. Using a variable exon-specific mAb, it has been reported that expression of variable exon 6 (V6)- containing isoforms confers metastatic behavior to non- INTRODUCTION metastatic tumors of the rat in vivo (19, 20). In hu- CD44 is a transmembrane glycoprotein that is widely mans, the expression of V6 has also been correlated expressed on T and B lymphocytes, NK cells, mono- with metastatic behavior in some tumors (21 – 28; re- cytes, and epithelial cells (reviewed in 1, 2). The wide viewed in 29, 30). Furthermore, an antibody against distribution of CD44 expression in addition to the the V6 product of CD44 was found to modify immune highly regulated patterns of expression observed dur- function in vivo in the rat (31). ing functional maturation and cell differentiation, es- Previously, PCR methodologies have been used to pecially among lymphoid cells, suggests that CD44 may demonstrate that the expression of mRNA of variable play a fundamental role in cell – cell and/or cell – matrix exon-containing isoforms of CD44 is regulated in a tis- sue-specific manner in the mouse (17, 32). In the pres- ent study, we have carried out an analysis of variable 1 To whom correspondence and reprint requests should be ad- exon expression at the level of protein products using dressed at Experimental Immunology Branch, National Cancer In- a panel of monoclonal antibodies against variable exon- stitute, National Institutes of Health, Building 10, Room 4B17, 10 Center Drive MSC 1360, Bethesda, MD 20892-1360. specific epitopes of mouse CD44. For this purpose, fu- 22 0008-8749/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. AID CI 1056 / 6c1a$$$241 01-31-97 20:57:28 cia